The present invention relates to the use of multiphoton excitation in conjunction with optical biopsy needles and endoscopes.
Many crucial biological functions are mediated or accomplished by biomolecules and tissue structures that are intrinsically fluorescent. As a result, there is an opportunity to diagnose and study important biological events by measuring and localizing the spectra and tissue fluorescence emission. To investigate in vivo internal processes and structures in large organisms, such as human beings and agricultural animals, endoscopic procedures which penetrates body cavities or even solid tissue may be required. Endoscopy video imaging in body cavities ordinarily utilizes back-scattered white light applied through the endoscope to form a low-resolution color image of the internal surfaces of these cavities. Physicians often use the changes in shapes and changes in local apparent color (which are often due to changes in blood distribution) to recognize disease states, such as malignant tumors or inflammation. Unfortunately, these clues are frequently not sufficient, especially for detection of the early onset of disease. Diagnostic improvements have been made by quantitative measurements of the light scattering and of tissue fluorescence emission. Ordinarily, the light required to excite the fluorescence of tissue is delivered through an optical fiber or fiber bundle that is inserted through a small tube built into the endoscopic pipe to accommodate a mechanical biopsy wire. Small optical fibers or fiber bundles can be passed easily through the same tube. Some of the strongest tissue fluorescence usually seen in this procedure are due to NADH (nicotinamide adenine dinucleotide) and to collagen structures. Their fluorescence is excited by absorption of ultraviolet light of about 300 to 400 nm wavelength corresponding to photon energies of around 3 to 4 eV or sometimes slightly longer wavelength visible light.
A first problem is that this ultraviolet light is strongly absorbed by hemoglobin and oxyhemoglobin in the blood, which are not fluorescent, so that penetration of the illumination into the tissue depends on their concentration and distribution.
A second problem is that the illumination exiting the optical fibers into tissue fans out at an included angle determined by the numerical aperture (NA) of the optical fiber. Small lenses can focus the spread so that the light first converges to a focus but it then fans out beyond the focal plane. (Typically, the NA is about 0.2 and the included cone angle isxcx9c17xc2x0.) This angular spreading is a problem, because roughly equal total amounts of fluorescence are excited in every spherical section at each distance from the end of the fiber until attenuated by absorption. This effect is schematically illustrated in FIG. 1. Fluorescence excitation is similarly spread out. Scattering does not attenuate the fluorescence excitation but does distribute it even more broadly. Consequently, the volume observed is ill defined with its practical limits depending also on blood distribution and light scattering. It should be noted that these problems tend to persist even if lenses focus the illumination and/or prisms and mirrors deflect the light for side viewing.
The present invention is directed to overcoming these deficiencies in the art.
One aspect of the present invention relates to a method of detecting disease within a particular tissue of a plant or animal. This method involves activating the particular plant or animal tissue by application of radiation through at least one optical fiber under conditions effective to promote a simultaneous multiphoton excitation of the particular plant or animal tissue and to emit an intrinsic fluorescence characteristic. The intrinsic fluorescence characteristic is compared to fluorescence emitted by exciting healthy tissue of the particular plant or animal under the same conditions used to carry out the activating step. The particular tissue of a plant or animal where the intrinsic fluorescence characteristic differs from the fluorescence, emitted by exciting healthy tissue of the particular plant or animal under the same conditions used to carry out such activity, is then identified as potentially diseased.
Another aspect of the present invention involves a method of producing an image of an internal surface of a particular tissue within a plant or animal. In this method, the particular plant or animal tissue is activated with radiation applied through an optical fiber under conditions effective to promote a simultaneous multiphoton excitation of the internal surface of the particular tissue within the plant or animal and produce an autofluorescence. The autofluorescence is collected to produce an image of the internal surface.
The present invention also relates to a method of detecting and localizing fluorescence within a subject by applying radiation to an internal region of the subject through at least one optical fiber. Each fiber terminates in a tip proximate to the internal region. Radiation is applied under conditions effective to cause simultaneous multiphoton absorption of fluorophore molecules within the internal region and, as a result, fluorescent excitation proximate to the tip of the at least one optical fiber.
Another embodiment of the present invention relates to a method of detecting and localizing fluorescence within a body of penetrable material by applying radiation to an internal region of the body of penetrable material through at least one optical fiber. Each fiber terminates in a tip proximate to the internal region. Radiation is applied under conditions effective to cause simultaneous multiphoton absorption of fluorophore molecules within the internal region and, as a result, fluorescent excitation proximate to the tip of the at least one optical fiber.
The present invention utilizes multiphoton absorption to excite autofluorescence of tissue with good spatial resolution in order to recognize disease by autofluorescence spectroscopy. The most useful tissue fluorescence for this purpose is most likely to require absorption of ultraviolet energies for excitation. Multiphoton excitation provides the added convenience of infrared illumination to provide the necessary excitation energy by simultaneous absorption of two or more photons by the fluorescent molecules or structures.
The same advantages described above for internal multiphoton excitation of fluorescence spectroscopy through optical fibers penetrating a body cavity or tissue can be advantageously applied to probe other penetrable materials that are, or can be made, fluorescent. Some examples are: food products, polymeric structures or porous media.
One important advantage of multiphoton excitation is that the illumination is not absorbed by hemoglobin and myoglobin and, in fact, not strongly absorbed by any other common tissue components. Scattering of infrared light by tissue is also significantly less than scattering by ultraviolet wavelengths. The principal advantage of multiphoton excitation, however, for endoscopic fluorescence spectroscopy is that the effective focal volume within which fluorescence is strongly excited is well defined and highly localized (FIG. 2). The reason for this is that the rate of two-photon excitation of fluorescence is proportional to the square of the illumination intensity. For higher multiphoton processes, the power law exponent of the intensity is larger, cubic for three-photon excitation for example. In multiphoton laser scanning microscopy, this higher power law feature makes possible three-dimensional resolution without generating out-of-focus fluorescence that would have to be excluded by confocal spatial filtering.
Nearly the same illumination conditions are also suitable for generation of second and third harmonic generation in certain suitable tissues. The second and third harmonics are generated, respectively, at exactly one half and one third of the laser illumination wavelengths and can be used with the intrinsic fluorescence to help characterize the tissue.
An analogous advantage applies to fluorescence multiphoton excited by laser light transmitted through an optical fiber as in application to endoscopic tissue fluorescence. Although the illumination intensity fans out just as for one-photon excitation with roughly equal total power at each value of radius from the end of the fiber or focal plane of any focusing lens, the excited fluorescence does not follow this fanned out illumination. In the case of two-photon excitation through a single mode optical fiber, the distribution of fluorescence is localized near the fiber tip in a shape resembling a candle flame beginning at the fiber tip. This effective focal volume is defined by the spatial distribution of the square of the illumination intensity for two-photon excitation. The square of the illumination intensity falls off roughly as the reciprocal fourth power of the distance from the fiber tip so that the fluorescence excitation is localized. Significant fluorescence is emitted only from this limited volume where the square of the excitation intensity is large, as illustrated in FIG. 3. Lenses, prisms, etc. can be used to shift the effective focal volume beyond the end of the optical fiber and/or to provide a side-looking orientation without losing the advantages of multiphoton excitation.
This effect provides a well-defined focal volume for the fluorescence excitation and allows useful spatial resolution which is sharp enough to resolve important anatomical structures. For example, in the colon, about 5 distinct layers should be distinguishable. At the surface of the endothelium, an array of crypts covers the area and is terminated in a cellular layer that closes the bottoms of the crypts, followed by several more layers including smooth muscle and connective tissue for a total of about 0.5 mm. These layers are readily resolved by multiphoton laser scanning microscopy exciting the intrinsic tissue fluorescence, which differs from layer to layer. Such layers and their perturbations by disease near the surface are thus distinguishable in the intact tissue by endoscopic spectroscopy of the tissue fluorescence with sufficient spatial resolution as provided by multiphoton excitation.
The multiphoton excitation of the present invention allows accurate spatial discrimination and permits quantification of fluorescence from small volumes whose locations are defined in three dimensions. This is especially important in cases where thicker layers of cells are to be studied. In this case, the fiber can penetrate the tissue to observe and resolve the multiphoton excited fluorescence of deeper layers, thus providing optical biopsy in situ. Furthermore, multiphoton excitation greatly reduces the background fluorescence and scattering artifacts.